Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.     

The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta vollenweideri)

Social insects show a variety of temperature-guided behaviors. Depending on whether heat reaches the sensillum via air movements (convective heat) or as radiant heat, specific adaptations of thermo-sensitive sensilla are expected. In the present study the morphology and the physiology of thermo-sensitive peg-in-pit sensilla (S. coeloconica) of the leaf-cutting ant Atta vollenweideri were investigated. S. coeloconica are located predominantly in a single cluster on the apical antennomere, and connect to the outside through a small aperture. The sensory peg is double-walled, embedded in a chamber and innervated by three unbranched dendrites. Using tungsten electrodes, activity of the sensory neurons was measured. In most cases, the neuron with the largest spike amplitude responds to changes in air temperature (convective heat) as well as to radiant heat. In response to a drop in air temperature, the neuron shows a phasic-tonic response followed by a complete adaptation within 1 min (cold-sensitive neuron). Based on their morphology and physiology, it is suggested that the S. coeloconica are involved in the recently described thermal orientation behavior of A. vollenweideri leaf-cutting ants.     

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

PI3K-dependent lysosome exocytosis in nitric oxide-preconditioned hepatocytes

We investigated the signal mediators and the cellular events involved in the nitric oxide (NO)-induced hepatocyte resistance to oxygen deprivation in isolated hepatocytes treated with the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])diazen-1-ium-1,2-diolate (NOC-9). NOC-9 greatly induced PI3K activation, as tested by phosphorylation of PKB/Akt. This effect was prevented by either 1H-(1,2,4)-oxadiazolo-(4,3)-quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC), or KT5823, an inhibitor of cGMP-dependent kinase (cGK), as well as by farnesyl protein transferase inhibitor, which blocks the function of Ras GTPase. Bafilomycin A, an inhibitor of the lysosome-type vacuolar H+-ATPase, cytochalasin D, which disrupts the cytoskeleton-dependent organelle traffic, and wortmannin, which inhibits the PI3K-dependent traffic of lysosomes, all abolished the NOC-9-induced hepatocyte protection. The treatment with NOC-9 was associated with the PI3K-dependent peripheral translocation and fusion with the plasma membrane of lysosomes and the appearance at the cell surface of the vacuolar H+-ATPase. Inhibition of sGC, cGK, and Ras, as well as the inhibition of PI3K by wortmannin, prevented the exocytosis of lysosomes and concomitantly abolished the protective effect of NOC-9 on hypoxia-induced pHi and [Na+]i alterations and cell death. These data indicate that NO increases hepatocyte resistance to hypoxic injury by activating a pathway involving Ras, sGC, and cGK that determines PI3K-dependent exocytosis of lysosomes.     

Clinical forms of Trypanosoma cruzi infected individuals in the chronic phase of Chagas disease in Puebla, Mexico

In Mexico, despite the relatively high seroprevalence of Trypanosoma cruzi infection in humans in some areas, reported morbidity of Chagas disease is not clear. We determined clinical stage in 71 individuals seropositive to T. cruzi in the state of Puebla, Mexico, an area endemic for Chagas disease with a reported seroprevalence of 7.7%. Diagnosis of Chagas disease was made by two standardized serological tests (ELISA, IHA). Individuals were stratified according to clinical studies. All patients were submitted to EKG, barium swallow, and barium enema. Groups were identified as indeterminate form (IF) asymptomatic individuals without evidence of abnormalities (n = 34 cases); those with gastrointestinal alterations (12 patients) including symptoms of abnormal relaxation of the lower esophageal sphincter and absent peristalsis in the esophageal body, grade I megaesophagus, and/or megacolon; patients with clinical manifestations and documented changes of chronic Chagas heart disease who were subdivided as follows: mild (8 patients)--mild electrocardiographic changes of ventricular repolarization, sinus bradychardia); moderate (6 patients)--left bundle branch block, right bundle branch block associated with left anterior fascicular block); severe (8 patients)--signs of cardiomegaly, dilated cardiomyopathy); and the associated form (3 cases) that included presence of both cardiomyopathy and megaesophagus. These data highlight the importance of accurate evaluation of the prevalence and clinical course of Chagas disease in endemic and non-endemic areas of Mexico.